The aim of the present study was to investigate toll‐like receptor‐4 (TLR4) signalling at different stages of non‐alcoholic fatty liver disease (NAFLD) induced by high fat high fructose (HFHFr) diet in mice. TLR4 wild type (WT) and mutant (TLR4mut) mice were fed with either standard chow (SC) or HFHFr diet for different periods of time from 4 to 16 weeks. The liver pathological characteristics and functions were assessed. Simple steatosis, steatohepatitis and hepatic fibrosis occurred sequentially in week 4, 8 and 16 in the WT mice fed with HFHFr. The expression levels of TLR4, MyD88, IRF3 and IRF7 started to increase at the time course of week 4, peaked at week 8, and then declined to basal levels at week 16. This alteration pattern was consistent with the changes of inflammation in the livers revealed by H&E staining. However, lipid accumulation, inflammation and fibrosis in the livers of TLR4mut mice given HFHFr diet were significantly alleviated. In addition, the expression of activin‐A in TLR4‐WT mice fed with HFHFr diet increased at week 16. Our dada suggest that TLR4 signaling mediates non‐alcoholic steatohepatitis before fibrosis, and activin‐A is subsequently involved in NAFLD. This article is protected by copyright. All rights reserved.